Project Summary About 70% of patients with metastatic breast cancer develop osteolytic metastases (OM) leading to life threatening health complications. Bisphosphonates (BPs) are prescribed to treat OM and there is evidence that BPs induce tumor-cell apoptosis at the metastatic site. However, their direct anti-tumor effect remains incon- sistent in in vivo clinical research. This might be due to the high doses required to achieve meaningful anti- tumor effects, as BPs present short blood circulation times. Recent clinical trials show that combining BPs with endocrine or chemotherapy improves patient survival. Thus, the development of robust bone-selective multi- drug delivery systems for the co-administration of these drugs could result in improved outcomes. The pro- posed pilot project will coordinate clinically relevant BPs with bioactive metals to form porous nano-structures denominated as BP-based biocompatible coordination complexes (nano-pBioCCs). It is hypothesized that the resulting nano-pBioCCs could be potentially employed to treat and prevent OM, if therapeutic quantities of en- docrine or chemotherapeutic drugs could be encapsulated and later controlled-released at the metastatic site. To test the hypothesis we propose the following specific aims. Aim 1 involves the synthesis and characteriza- tion of porous nano-pBioCCs. We will employ BPs from the two most potent generations, alendronic (2nd gen- eration) and risedronic and zoledronic acids (both 3rd generation), which present high anti-resorptive potencies. Aim 1 will assess the ability of BPs to form porous nano-pBioCCs with bioactive metals, the stability of the pBi- oCCs under simulated physiological conditions, and the ability of nano-pBioCCs to bind selectively to the bone microenvironment. In Aim 2, we will assess the capacity of nano-pBioCCs to encapsulate and controlled- release three model drugs, palbociclib (PAL), letrozole (LET), and 5-fluorouracil (5-FU). These drugs do not need to be metabolized and present molecular sizes that enable their encapsulation. PAL treats HR+ and HER2- metastatic breast cancer in combination with an aromatase inhibitor (AI), often LET. However, LET and other AIs are associated with increasing risks of osteoporotic fractures and bone loss. Unlike the commonly used doxorubicin, 5-FU does not induce osteolytic bone damage. BPs prevent treatment-related bone loss and skeletal complications of metastatic diseases. Yet, adverse effects are associated with their high doses. Aim 2 will demonstrate the utility of nano-pBioCCs to release encapsulated drugs. In Aim 3 we will assess the cyto- toxicity and effectiveness of nano-pBioCCs using cocultures of human breast cancer cells (MCF7 and MDA- MB-231) with normal osteoclasts-like (2T-110) or osteoblasts-like (hFOB1.19) cell lines in a silk-based in vitro model, that mimics the 3D microenvironment of bone-breast cancer. The realization this pilot study will pave the way to engineer robust nano-formulations that could be used as bone-selective multidrug delivery sys- tems relevant to the treatment of OM with broader applications to other bone-related diseases.